Preliminary studies have determined that it is possible to maintain and grow heart muscle cells, isolated from adult rats, in long-term culture. These cells are isolated from adult rat heart by retrograde perfusion via the aorta with buffer containing 1 mg/ml of collagenase. Freshly isolated cells are striated and cylinder-shaped, typical of intact ventricular cardiac muscle. When placed in culture these cells round-up and lose their cross-striation. They then send out pseudopod-like processes and eventually spread out and become entirely flattened. With increasing time in culture, organized bands of myofibrils reappear. The major objective of this research project is to systematically investigate the conditions which are required to maintain these cells in long-term culture and to characterize them both morphologically and biochemically. Various growth factors, hormones and chemicals will be added to the culture medium in order to try to improve the growth of these cells. At various times after placing them in culture they will be examined with the light microscope and with the transmission and scanning electron microscopes in order to assess their external and internal morphology. Biochemical characterization will include measurements of the ability of the cells to synthesize DNA, protein and myosin at various time periods during culture. Myosin isozymes and the activities of several DNA enzymes and creatine kinase will be measured to assess the degree of dedifferentiation and redifferentiation of these cells in culture. Additional studies will be done to determine the metabolic activity of the cells and how it compares to freshly isolated cells and to intact tissue. Establishment and characterization of adult cardiac myocytes in long-term cell culture will provide a valuable system to study the molecular biology, biochemistry, physiology and pharmacology of the adult heart cell.